1. Field of the Invention
The present invention relates to a novel brazzein multiple variants having increased sweetness, and a production method for the same.
2. Discussion of Related Art
White sugar (refined sugar) is a type of saccharide, and more specifically, is a disaccharide referred to as a kind of saccharose (a chemical term referring to sugar) composed of a simple carbohydrate called “sucrose”. Sugar has been frequently used as a sweetener for a long period of time. However, the World Health Organization (WHO) has proposed a recommendation to limit the consumption of sugar to 10% of the current level because of health problems caused by sugar, and state governments of the United States have prohibited in selling foods including sugar as a major ingredient and beverages including a high content of sugar. Further, in Korea, the National Obesity Taskforce has been organized to announce a policy that sugar manufacturers need to include warning labels about sugar risks on their products, and is scheduled to regulate advertisements for foods including sugar exceeding a standard sugar content after 2010. Consequently, there is a need for the emergence of a new sweetener that may be substituted for sugar. In 1879, Ira Remsen from USA and Constantin Fahlberg from Germany discovered saccharin, which is 500 times sweeter than sugar. Saccharin has an advantage in that saccharin does not break down in the human body and is excreted from the human body, but has sparked a controversy that saccharin is a carcinogenic substance. Finally, although saccharin was proven to be harmless to the human body, saccharin is still scarcely used due to the disadvantage of the bitter aftertaste of saccharin. In 1937, the University of Illinois in USA found that sodium cyclohexylsulfamate has a sweet taste. With the trade name cyclamate, sodium cyclohexylsulfamate was first used in the beginning of 1950, and became popular on the global sweetener market in the 1960s. However, as sodium cyclohexylsulfamate was proven to be a carcinogenic substance, sodium cyclohexylsulfamate has been completely prohibited since the 1970s in Korea. An artificial sweetener most widely used in recent years is aspartame that was discovered in 1965 by James M. Schlatter. Aspartame has a sugar content about 180 to 200 times that of sugar. Aspartame is included in a majority of currently commercially available diet beverages, and thus produces phenylalanine during the metabolism process when aspartame is ingested into the human body. Consequently, aspartame has a disadvantage in that phenylketonuric patients who are congenitally deficient in a specific enzyme (phenylalanine hydroxylase) to break down phenylalanine cannot use aspartame.
There has been continuous research conducted to develop not only artificial sweeteners but also natural sweeteners, and as a result, a material referred to as stevioside was found to be present in the leaves of a perennial plant (Stevia rebaudiana) in the Asteraceae, which is classified as an herb. The natives living in the border between Paraguay and Brazil have used this material as a sweetener for over 400 years. In Korea, stevioside is sometimes added to a traditional distilled liquor called “soju” and is 200 times as sweet as sugar. Meanwhile, increasing attention has been recently paid to a sweetener protein extracted from a tropical fruit, and Thaumatin is a protein included in the fruit of a perennial plant (Thaumatococcus daniellii) referred to as a miracle fruit in Western Africa, and is 2,000 to 3,000 times as sweet as sugar. Monellin is a protein obtained from the fruit of a vine plant referred to as a serendipity berry growing in the rain forest area of Africa, and is 3,000 times as sweet as sugar. However, it is difficult to culture the serendipity berry and also to extract monellin from the fruit of the serendipity berry. Moreover, monellin has a disadvantage in that monellin has low thermal stability, and loses its 3-dimensional protein structure and thus does not show sweetness when being heat-treated in a food processing process. In order to overcome these disadvantages, there has been research conducted to enhance the thermal stability of the monellin using a protein engineering technique.
Meanwhile, brazzein is a sweetener protein firstly extracted from the fruit of Pentadiplandra brazzeana (Baillon) growing in West Africa [Ming et al., FEBS Letters, 355: 106-108, 1994]. Brazzein shows sweetness about 500 to 2,000 times or more that of sucrose [Jin et al., Chem. Senses. 28: 491-498, 2003], and is divided into two types: a major type and a minor type. The major type accounting for a majority of brazzein extracted from the plant has 54 amino acids including a pyroglutamic acid residue bound to an amino-terminal site. On the other hand, the minor type of brazzein has 53 amino acid residues without a pyroglutamic acid residue bound to an amino-terminal site, and shows stronger sweetness, about twice that of the major type of brazzein [Assadi-Porter et al., Arch., Biochem. Biophys. 376: 259-265, 2000]. Brazzein has a molecular weight of about 6.5 kDa, which is the smallest among the sweetener proteins, and is a monomer composed of one kind of subunit. Brazzein consists of a single polypeptide and is composed of one a-helix and two β-pleated sheets. Brazzein has very high thermal stability since brazzein has 8 cysteine residues to form 4 disulfide bonds in the molecule. In addition, brazzein shows very high solubility and pH stability in water [Gao et al., Int. J. Biol. Macromol. 24: 351-359, 1999].
U.S. Pat. No. 6,274,707 B1 and Assadi-Porter et al. (Assadi-Porter et al., Arch. Biochem. Biophys. [0006] 376: 259-265, 2000) describe a method of producing recombinant brazzein using a genetic engineering method by which the aforementioned brazzein is produced in Escherichia coli, and disclose the method including: synthesizing a gene encoding brazzein, inserting the gene into a recombinant vector containing a SNase gene to produce a new transformation vector, introducing the transformation vector into E. coli, and finally expressing and purifying a fusion protein linked with the SNase. However, since the brazzein fused and expressed with the SNase produces an insoluble inclusion body, and the insoluble inclusion body is refolded and separated and purified by a method of removing SNase and methionine (Met) using cyanobromide (CNBr), there is a disadvantage in that the method is technically complex and difficult so that it is very difficult to commercialize the recombinant brazzein by mass production. Accordingly, the present inventors have conducted prior research to solve the disadvantages of the existing researches, and registered a patent for a polynucleotide including an E. coli pelB signal sequence and a brazzein gene and a production method for brazzein using the same (Korean Patent No. 809100). Furthermore, in order to search for a natural sweetener having high thermal stability and showing excellent sweetness, the present inventors filed patent applications (Korean Patent Application Nos. 2007-0117013, 2008-0019008, and 2010-0016660, and International Patent Application No. PCT/KR2009/04855) for a production method for variants and multiple variants of amino acids at certain positions which are expected not to affect a structure in an amino acid constituting brazzein.
Throughout the present specification, a plurality of papers and patent documents are referenced, and citations thereof are indicated. The disclosure of each of the cited papers and patent documents is incorporated herein by reference in its entirety to describe the level of the technical field to which the present invention pertains and the content of the present invention more apparently.